Use of whole genome amplification to rescue DNA from plasma samples

While DNA of good quality and sufficient amount can be obtained easily from whole blood, buccal swabs, surgical specimens, or cell lines, these DNA-rich sources are not always available. This is particularly the case in studies for which biological specimens were collected when genotyping assays were not widely available. In those studies, serum or plasma is often the only source of DNA. Newly developed whole genome amplification (WGA) methods, based on phi29 polymerase, may play a significant role in recovering DNA in such instances. We tested a total of 528 plasma samples kept in storage at -40 degrees C for approximately 10 years for 8 single nucleotide polymorphisms (SNPs) using the 5' exonuclease (TaqMan) assay. These specimens yielded undetectable levels of DNA following extraction with an affinity column but produced an average 52.7 microg (standard deviation of 31.2 microg) of DNA when column-extracted DNA was used as a template for WGA. This increased the genotyping success rate from 54% to 93%. There were only 3 disagreements out of 364 paired genotyping results for pre- and post-WGA DNAs, indicating an error rate of 0.82%. These results are encouraging for expanding the use of poor DNA resources in genotyping studies.     

An Improved Field Method to Obtain DNA for Individual Identification From Wolf Scat

ABSTRACT Sampling of feces for genetic studies of wild populations can be problematic because of the low quality and quantity of template DNA obtained. We used cotton swabs in the field to isolate the mucous layer on the surface of fresh wolf (Canis lupus, C. lycaon, and their hybrids) scats followed by immediate preservation, and compared microsatellite genotyping of DNA from these fresh field swabs (FS) to that of previously frozen laboratory swabs (LS). In single polymerase chain reactions (PCRs) of 2 multiplexes, amplification at 8 loci was higher in the FS samples (FS = 50%, LS = 15%; P = 0.02) because proportion, quantity, and quality of large fragment wolf nuclear DNA from these samples was greater (2.5–25%, 6.25–62.5 ng/swab, 35% amplified at 1,000 base pairs [bp]) than from the LS samples (1.9%–10%, 4.7–25 ng/swab, 10% amplified at 1,000 bp). Paired blood and fresh field-swabbed samples had identical genotypes. In 84 multiplex PCRs we found no evidence of allelic dropout associated with low template quality or quantity. We conclude that field swabbing of fresh wolf scat facilitates field storage and reduces the need for multiple amplifications at single microsatellite loci, thereby reducing the genotyping costs for wildlife projects that use noninvasive samples.     

Genotyping of Single Nucleotide Polymorphisms in DNA Isolated from Serum Using Sequenom MassARRAY Technology

Background

Large epidemiologic studies have the potential to make valuable contributions to the assessment of gene-environment interactions because they prospectively collected detailed exposure data. Some of these studies, however, have only serum or plasma samples as a low quantity source of DNA.

Methods

We examined whether DNA isolated from serum can be used to reliably and accurately genotype single nucleotide polymorphisms (SNPs) using Sequenom multiplex SNP genotyping technology. We genotyped 81 SNPs using samples from 158 participants in the NYU Women’s Health Study. Each participant had DNA from serum and at least one paired DNA sample isolated from a high quality source of DNA, i.e. clots and/or cell precipitates, for comparison.

Results

We observed that 60 of the 81 SNPs (74%) had high call frequencies (≥95%) using DNA from serum, only slightly lower than the 85% of SNPs with high call frequencies in DNA from clots or cell precipitates. Of the 57 SNPs with high call frequencies for serum, clot, and cell precipitate DNA, 54 (95%) had highly concordant (>98%) genotype calls across all three sample types. High purity was not a critical factor to successful genotyping.

Conclusions

Our results suggest that this multiplex SNP genotyping method can be used reliably on DNA from serum in large-scale epidemiologic studies.     

Multiple Displacement Amplification for Generating an Unlimited Source of DNA for Genotyping in Nonhuman Primate Species

We evaluated a whole genome amplification method—multiple displacement amplification (MDA)—as a means to conserve valuable nonhuman primate samples. We tested 148 samples from a variety of species and sample sources, including blood, tissue, cell-lines, plucked hair and noninvasively collected semen. To evaluate genotyping success and accuracy of MDA, we used routine genotyping methods, including short tandem repeat (STR) analysis, denaturing gradient gel electrophoresis (DGGE), Alu repeat analysis, direct sequencing, and nucleotide detection by tag-array minisequencing. We compared genotyping results from MDA products to genotypes generated from the original (non-MD amplified) DNA samples. All genotyping methods showed good results with the MDA products as a DNA template, and for some samples MDA improved genotyping success. We show that the MDA procedure has the potential to provide a long-lasting source of DNA for genetic studies, which would be highly valuable for the primate research field, in which genetic resources are limited and for other species in which similar sampling constraints apply.     

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes¹, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes^2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.     

Canine DNA subjected to whole genome amplification is suitable for a wide range of molecular applications

Molecular and genetic studies of canine disease phenotypes can be limited by the amount of DNA available for analysis. New methods have been developed to amplify the genomic DNA of a species producing large quantities of DNA from small starting amounts. Whole genome amplification (WGA) of DNA is now being used in human studies, although this technique has not been applied extensively in veterinary research. We evaluated WGA of canine DNA for suitability in a range of molecular tests. DNA from 93 canine blood extracted and 18 buccal swab samples was subjected to WGA using the GenomiPhi kit (Amersham). Genomic DNA was compared with WGA product using a range of techniques, including reference strand-mediated conformation analysis, denaturing high-performance liquid chromatography analysis, microsatellite genotyping, direct DNA sequencing, and single nucleotide polymorphism allelic discrimination. All samples amplified well, giving an average yield of 3 mug of DNA from 2.5 ng of starting material. Extremely high levels of experimental reproducibility and concordance were observed between source and WGA DNA samples for all analyses used: greater than 95% for blood extracted DNA and greater than 80% for buccal swab DNA. These studies clearly demonstrate the usefulness of WGA of canine DNA as a means of increasing DNA quantities for canine studies. This technique will have major implications for future veterinary research.     

Molecular genetic analysis of the polymorphic apolipoprotein E in modern and ancient human DNA samples

In this report, methodical bases for the molecular genetic analysis of the three common apolipoprotein E alleles APOE*2, APOE*3 and APOE*4 in DNA isolated from ancient human skeletal remains are described. Considering that ancient DNA target regions for amplification are generally quite small, the detection method is based on short amplification products in the range from 71 bp to 75 bp. The applicability of the modified method for APOE genotyping was examined in modern human DNA samples.     

Allelic dropout from a high-quality DNA source

Allelic dropouts are an important source of genotyping error, particularly in studies using non-invasive sampling techniques. This has important implications for conservation biology, as an increasing number of studies are now using non-invasive techniques to study rare species or endangered populations. Previously, allelic dropout has typically been associated with PCR amplification of low quality/quantity template DNA. However, in this study we recorded high levels of allelic dropout (21–57%) at specific loci amplified from a high quality DNA (63.1 ± 7.8 ng/μl) source in the red fox (Vulpes vulpes). We designed a series of experiments to identify the sources of error. Whilst we were able to show that the best method to identify allelic dropout was the dilution of template DNA prior to PCR amplification, our data also showed two specific patterns: (1) allelic dropouts occurred at specific loci; (2) allelic dropouts occurred at specific pair-wise combinations of alleles. These patterns suggest that mechanisms other than low quantity template DNA are responsible for allelic dropout. Further research on the causes of these patterns in this and other studies would further our understanding of genotyping errors and would aid future studies where allelic dropout may be a serious issue.     

Saliva samples are a viable alternative to blood samples as a source of DNA for high throughput genotyping

ABSTRACT: BACKGROUND: The increasing trend for incorporation of biological sample collection within clinical trials requires sample collection procedures which are convenient and acceptable for both patients and clinicians. This study investigated the feasibility of using saliva-extracted DNA in comparison to blood-derived DNA, across two genotyping platforms: Applied Biosystems Taqman TM and Illumina Beadchip TM genome-wide arrays. METHOD: Patients were recruited from the Pharmacogenetics of Breast Cancer Chemotherapy (PGSNPS) study. Paired blood and saliva samples were collected from 79 study participants. The Oragene DNA Self-Collection kit (DNAgenotek(R)) was used to collect and extract DNA from saliva. DNA from EDTA blood samples (median volume 8 ml) was extracted by GenProbe, Livingstone, UK. DNA yields, standard measures of DNA quality, genotype call rates and genotype concordance between paired, duplicated samples were assessed. RESULTS: Total DNA yields were lower from saliva (mean 24 ug, range 0.2-52 ug) than from blood (mean 210 ug, range 58-577 ug) and a 2-fold difference remained after adjusting for the volume of biological material collected. Protein contamination and DNA fragmentation measures were greater in saliva DNA. 78/79 saliva samples yielded sufficient DNA for use on Illumina Beadchip arrays and using Taqman assays. Four samples were randomly selected for genotyping in duplicate on the Illumina Beadchip arrays. All samples were genotyped using Taqman assays. DNA quality, as assessed by genotype call rates and genotype concordance between matched pairs of DNA was high (>97%) for each measure in both blood and saliva-derived DNA. CONCLUSION: We conclude that DNA from saliva and blood samples is comparable when genotyping using either Taqman assays or genome-wide chip arrays. Saliva sampling has the potential to increase participant recruitment within clinical trials, as well as reducing the resources and organisation required for multicentre sample collection.     

DNA sampling from eggshell swabbing is widely applicable in wild bird populations as demonstrated in 23 species

There is increasing interest in noninvasive DNA sampling techniques. In birds, there are several methods proposed for sampling DNA, and of these, the use of eggshell swabbing is potentially applicable to a wide range of species. We estimated the effectiveness of this method in the wild by sampling the eggs of 23 bird species. Sampling of eggs was performed twice per nest, soon after the clutch was laid and again at the end of egg incubation. We genotyped DNA samples using a set of five conserved microsatellite markers, which included a Z-linked locus and a sex-typing marker. We successfully collected avian DNA from the eggs of all species tested and from 88.48% of the samples. In most of the cases, the DNA concentration was low (ca. 10 ng/μL). The number of microsatellite loci amplified per sample (0-5) was used as a measure of the genotyping success of the sample. On average, we genotyped 3.01 ± 0.12 loci per sample (mean ± SE), and time of sampling did not seem to have an effect; however, genotyping success differed among species and was greater in those species that used feather material for lining their nest cups. We also checked for the occurrence of possible genotyping errors derived from using samples with very low DNA quantities (i.e. allelic dropout or false alleles) and for DNA contamination from individuals other than the mother, which appeared at a moderate rate (in 44% of the PCR replicates and in 17.36% of samples, respectively). Additionally, we investigated whether the DNA on eggshells corresponded to maternal DNA by comparing the genotypes obtained from the eggshells to those obtained from blood samples of all the nestlings for six nests of magpies. In five of the six magpie nests, we found evidence that the swab genotypes were a mixture of genotypes from both parents and this finding was independent of the time of incubation. Thus, our results broadly confirm that the swabbing of eggshells can be used as a noninvasive method for obtaining DNA and is applicable across a wide range of bird species. Nonetheless, genotyping errors should be properly estimated for each species by using a suite of highly polymorphic loci. These errors may be resolved by sampling only recently laid eggs (to avoid non-maternal DNA contamination) or by performing several PCR replicates per sample (to avoid allelic dropout and false alleles) and/or by increasing the amount of DNA used in the PCR through increasing the volume of the PCR or increasing the concentration of template DNA.     

Highly accurate SNP genotyping from historical and low‐quality samples

Historical and other poor‐quality samples are often necessary for population genetics, conservation, and forensics studies. Although there is a long history of using mtDNA from such samples, obtaining and genotyping nuclear loci have been considered difficult and error‐prone at best, and impossible at worst. The primary issues are the amount of nuclear DNA available for genotyping, and the degradation of the DNA into small fragments. Single nucleotide polymorphisms offer potential advantages for assaying nuclear variation in historical and poor‐quality samples, because the amplified fragments can be very small, varying little or not at all in size between alleles, and can be amplified efficiently by polymerase chain reaction (PCR). We present a method for highly multiplexed PCR of SNP loci, followed by dual‐fluorescence genotyping that is very effective for genotyping poor‐quality samples, and can potentially use very little template DNA, regardless of the number of loci to be genotyped. We genotyped 19 SNP loci from DNA extracted from modern and historical bowhead whale tissue, bone and baleen samples. The PCR failure rate was < 1.5%, and the genotyping error rate was 0.1% when DNA samples contained > 10 copies/µL of a 51‐bp nuclear sequence. Among samples with ≤ 10 copies/µL DNA, samples could still be genotyped confidently with appropriate levels of replication from independent multiplex PCRs.     

Single-copy nuclear DNA sequences obtained from noninvasively collected primate feces

Noninvasively collected primate feces have been shown to provide a useful source of mitochondrial DNA for sequencing and nuclear microsatellite DNA for size analysis. In this study, single-copy nuclear DNA sequences were obtained from noninvasively collected fecal samples of two species of wild tamarins, Saguinus fuscicollis and S. mystax, in the context of a project on the functional utility of color vision. Noninvasive genotyping of the X-linked opsin gene is important for future studies of selection and adaptation at this locus in a number of primate species. The wide range of techniques that can now be applied successfully to DNA extracted from feces introduces a broad spectrum of potential genetic studies that can be undertaken on primates, without the need for intrusive or invasive methods.     

Nondestructive DNA sampling from bumblebee faeces

Genetic studies provide valuable data to inform conservation strategies for species with small or declining populations. In these circumstances, obtaining DNA samples without harming the study organisms is highly desirable. Excrements are increasingly being used as a source of DNA in such studies, but such approaches have rarely been applied to arthropods. Bumblebees are ecologically and economically important as pollinators; however, some species have recently suffered severe declines and range contractions across much of Western Europe and North America. We investigated whether bumblebee faeces could be used for the extraction of DNA suitable for genotyping using microsatellite markers. We found that DNA could be extracted using a Chelex method from faecal samples collected either in microcapillary tubes or on filter paper, directly from captured individuals. Our results show that genotypes scored from faecal samples are identical to those from tissue samples. This study describes a reliable, consistent and efficient noninvasive method of obtaining DNA from bumblebees for use in population genetic studies. This approach should prove particularly useful in breeding and conservation programs for bumblebees and may be broadly applicable across insect taxa.     

Isolation of high-molecular-weight DNA from small samples of blood having nucleated erythrocytes,collected, transported,and stored at room temperature

Blood samples collected in the field for isolating DNA suitable for molecular analysis need special care in their storage and handling. In this article, we describe a simple method for the isolation of good-quality high-molecular-weight DNA that does not require low temperature conditions during collection, storage, and/or transportation of blood samples. This method involves smearing small aliquots of blood onto clean slides and air drying them at room temperature. The slides with blood smears can then be transported or stored at room temperature and still serve as a very good source of high-molecular-weight DNA. Genomic DNA from these samples can be extracted by organic phase separation (phenol-chloroform extraction) after lysis. The DNA thus obtained is of high quality and yields DNA fingerprints qualitatively similar to those prepared from corresponding control DNA isolated from frozen blood samples. Needing minimal facilities at field sites, the method is very convenient for conducting RFLP analysis of wild/field populations for demographic, behavioral, and ecologic studies.     

A test of the efficacy of whole‐genome amplification on DNA obtained from low‐yield samples

Conservation and population genetic studies are sometimes hampered by insufficient quantities of high quality DNA. One potential way to overcome this problem is through the use of whole genome amplification (WGA) kits. We performed rolling circle WGA on DNA obtained from matched hair and tissue samples of North American red squirrels (Tamiasciurus hudsonicus). Following polymerase chain reaction (PCR) at four microsatellite loci, we compared genotyping success for DNA from different source tissues, both pre‐ and post‐WGA. Genotypes obtained with tissue were robust, whether or not DNA had been subjected to WGA. DNA extracted from hair produced results that were largely concordant with matched tissue samples, although amplification success was reduced and some allelic dropout was observed. WGA of hair samples resulted in a low genotyping success rate and an unacceptably high rate of allelic dropout and genotyping error. The problem was not rectified by conducting PCR of WGA hair samples in triplicate. Therefore, we conclude that WGA is only an effective method of enhancing template DNA quantity when the initial sample is from high‐yield material.     

Noninvasive genetic analysis in birds: testing reliability of feather samples

Noninvasive samples are useful for molecular genetic analysis of free‐ranging animals. I tested whether moulted feathers collected in the field are a reliable source of DNA for genotyping microsatellite loci. I prescreened extracts for DNA quantity and, using only samples with higher amounts of DNA, obtained reliable genotyping results. Polymerase chain reaction (PCR) amplification success was higher from extracts of plucked feathers than moulted feathers. DNA quantity in larger feathers was higher than that in smaller feathers. This study clearly demonstrates that moulted feathers could be used for genetic studies in birds.     

Validation of a testosterone and dihydrotestosterone liquid chromatography tandem mass spectrometry assay: Interference and comparison with established methods

Testosterone (T) and its metabolite dihydrotestosterone (DHT) are androgens with different biologic profiles. T and DHT measurements are required for assessment of patients with ambiguous genitalia, hirsutism, during 5 alpha reductase treatment of prostate disorders, and new androgen formulations. Our laboratory has developed and validated a method to simultaneously measure serum T and DHT with liquid chromatography tandem mass spectrometry (LC-MS/MS) for use in a clinical chemistry laboratory. Analysis of sera from blood collected in tubes containing clot activator gave results of T that were fourfold higher than blood collected in plain tubes. Changing the ion pair selected for monitoring eliminated this interference by clot activators. Blood collected in fluoride-coated tubes gave serum T and DHT levels that were 20 and 15% lower, respectively than levels measured in blood collected in plain tubes (no additives). Addition of T enanthate to blood collected in plain tubes caused a dose related increase serum T levels due to the action of non-specific esterases in the red cells. This esterase activity could be avoided by using fluoride tubes for blood collection. Serum DHT levels were consistently lower when measured by LC-MS/MS versus radioimmunoassay. The differences were concentration dependent and the variance for the difference was large when serum DHT concentration was low. Celite chromatograph prior to radioimmunoassay reduced the differences between the two methods, thus confirming that higher levels of DHT obtained by immunoassays were probably due to interfering substances which were partially removed by Celite chromatography.     

Whole genome amplification of buccal cytobrush DNA collected for molecular epidemiology studies.

When cytobrush buccal cell samples have been collected as a genomic DNA (gDNA) source for an epidemiological study, whole genome amplification (WGA) can be critical to maintain sufficient DNA for genotyping. We evaluated REPLI-g WGA using gDNA from two paired cytobrushes (cytobush 'A' kept in a cell lysis buffer, and 'B' dried and kept at room temperature for 3 days, and frozen until DNA extraction) in a pilot study (n=21), and from 144 samples collected by mail in a breast cancer study. WGA success was assessed as the per cent completion/concordance of STR/SNP genotypes. Locus amplification bias was assessed using quantitative PCR of 23 human loci. The pilot study showed > 98% completion but low genotype concordance between cytobrush wgaDNA and paired blood gDNA (82% and 84% for cytobrushes A and B, respectively). Substantial amplification bias was observed with significantly lower human gDNA amplification from cytobrush B than A. Using cytobrush gDNA samples from the breast cancer study (n =20), an independent laboratory demonstrated that increasing template gDNA to the REPLI-g reaction improved genotype performance for 49 SNPs; however, average completion and concordance remained below 90%. To reduce genotype misclassification when cytobrush wgaDNA is used, inclusion of paired gDNA/wgaDNA and/or duplicate wgaDNA samples is critical to monitor data quality.     

A comparison between the concentration of prostaglandin F in human plasma and serum

The concentration of prostaglandin F (PGF) has been measured in serum and plasma samples prepared under different conditions from the antecubital vein blood of 4 non-pregnant and 7 pregnant women. Prostaglandin F concentrations were less than 41 pg/ml in 19 samples of serum or plasma prepared by centrifugation within 30 minutes of collection. When the blood was allowed to clot at room temperature for 24 hours, highly variable, but usually markedly increased concentrations of PGF (<30 - 3020 pg/ml) were found in the serum. Plasma obtained from blood which stood at 23°C for 24 hours contained undetectable amounts of PGF in 4 out of 6 samples and less than 75 pg/ml in the 2 remaining samples. Plasma and serum obtained from blood which stood at 4°C for 24 hours contained less than 45 pg PGF/ml. These results show that (i) incubation of blood at room temperature may markedly elevate concentrations of PGF in serum, (ii) plasma samples rather than serum should be used for measurements of PGF concentrations.     

Quantitation of DNA in buccal cell samples collected in epidemiological studies

Abstract

Buccal cell samples are increasingly used in epidemiological studies as a source of genomic DNA. The accurate and precise quantitation of human DNA is critical for the optimal use of these samples. However, it is complicated by the presence of bacterial DNA and wide inter-individual variation in DNA concentration from buccal cell collections. The paper evaluated the use of ultraviolet light (UV) spectroscopy, Höechst (H33258) and PicoGreen? as measures of total DNA, and real-time quantitative polymerase chain reaction (PCR) as a measure of human amplifiable DNA in buccal samples. Using serially diluted white blood cell DNA samples (at a concentration range of 300 to 0.5?ng µl^?1), UV spectroscopy showed the largest bias, followed by Höechst, especially for low concentrations. PicoGreen and real-time PCR provided the most accurate and precise estimates across the range of concentrations evaluated, although an increase in bias with decreasing concentrations was observed. The ratio of real-time PCR to PicoGreen provided a reasonable estimate of the percentage of human DNA in samples containing known mixtures of human and bacterial DNA. Quantification of buccal DNA from samples collected in a breast cancer case-control study by PicoGreen and real-time PCR indicated that cytobrush and mouthwash DNA samples contain similar percentages of human amplifiable DNA. Real-time PCR is recommended for the quantification of buccal cell DNA in epidemiological studies since it provides precise estimates of human amplifiable DNA across the wide range of DNA concentrations commonly observed in buccal cell DNA samples.